Letter | Published:

RIPK1 counteracts ZBP1-mediated necroptosis to inhibit inflammation

Nature volume 540, pages 124128 (01 December 2016) | Download Citation

Abstract

Receptor-interacting protein kinase 1 (RIPK1) regulates cell death and inflammation through kinase-dependent and -independent functions1,2,3,4,5,6,7. RIPK1 kinase activity induces caspase-8-dependent apoptosis and RIPK3 and mixed lineage kinase like (MLKL)-dependent necroptosis8,9,10,11,12,13. In addition, RIPK1 inhibits apoptosis and necroptosis through kinase-independent functions, which are important for late embryonic development and the prevention of inflammation in epithelial barriers14,15,16,17,18. The mechanism by which RIPK1 counteracts RIPK3–MLKL-mediated necroptosis has remained unknown. Here we show that RIPK1 prevents skin inflammation by inhibiting activation of RIPK3–MLKL-dependent necroptosis mediated by Z-DNA binding protein 1 (ZBP1, also known as DAI or DLM1). ZBP1 deficiency inhibited keratinocyte necroptosis and skin inflammation in mice with epidermis-specific RIPK1 knockout. Moreover, mutation of the conserved RIP homotypic interaction motif (RHIM) of endogenous mouse RIPK1 (RIPK1mRHIM) caused perinatal lethality that was prevented by RIPK3, MLKL or ZBP1 deficiency. Furthermore, mice expressing only RIPK1mRHIM in keratinocytes developed skin inflammation that was abrogated by MLKL or ZBP1 deficiency. Mechanistically, ZBP1 interacted strongly with phosphorylated RIPK3 in cells expressing RIPK1mRHIM, suggesting that the RIPK1 RHIM prevents ZBP1 from binding and activating RIPK3. Collectively, these results show that RIPK1 prevents perinatal death as well as skin inflammation in adult mice by inhibiting ZBP1-induced necroptosis. Furthermore, these findings identify ZBP1 as a critical mediator of inflammation beyond its previously known role in antiviral defence and suggest that ZBP1 might be implicated in the pathogenesis of necroptosis-associated inflammatory diseases.

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Acknowledgements

We thank Genentech and V. Dixit for Ripk3−/− mice, S. Akira for Zbp1−/− mice, and J. Han for lentiviral vectors. We thank B. Zevnik, P. Jankowski and S. Assenmacher at the CECAD Transgenic Core Facility for CRISPR/Cas9 mutagenesis in mouse zygotes and C. Uthoff-Hachenberg, J. Buchholz, E. Mahlberg and B. Kühnel for excellent technical assistance. Research reported in this publication was supported by funding from the ERC (grant agreement no. 323040) and the DFG (SFB829 and SFB670). J.L. was supported by a Humboldt research fellowship and C.K. was supported by a Humboldt research fellowship and an EMBO long-term fellowship.

Author information

Author notes

    • Juan Lin
    • , Snehlata Kumari
    •  & Chun Kim

    These authors contributed equally to this work.

Affiliations

  1. Institute for Genetics, Centre for Molecular Medicine (CMMC), and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany

    • Juan Lin
    • , Snehlata Kumari
    • , Chun Kim
    • , Trieu-My Van
    • , Laurens Wachsmuth
    • , Apostolos Polykratis
    •  & Manolis Pasparakis

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Contributions

J.L. designed and generated the Ripk1mRHIM mice. J.L. and C.K. analysed Ripk1mRHIM/mRHIM and RIPK1mRHIM/E-KO mice and performed genetic crosses to address the role of RIPK3 and ZBP1 in these mice. J.L. and C.K. carried out all immunoblots and immunoprecipitation experiments. S.K. generated and characterized RIPK1E-KO Zbp1−/− and FADDE-KO Zbp1−/− mice and made the initial discovery that ZBP1 is required for keratinocyte necroptosis in RIPK1E-KO mice. S.K. and T-M.V. conducted immunostainings and qRT–PCR assays in skin samples from RIPK1E-KO and RIPK1mRHIM/E-KO mice. A.P. generated the Ripk1fl/fl mice and L.W. generated Mlkl−/− mice. M.P. supervised the study, interpreted data and wrote the manuscript together with J.L., C.K. and S.K. J.L., C.K. and S.K. contributed equally and their order of appearance in the author list is random.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Manolis Pasparakis.

Reviewer Information Nature thanks H. Walczak and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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    Supplementary Information

    This file contains the uncropped blot scans.

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DOI

https://doi.org/10.1038/nature20558

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